Role Of P38 MAP Kinase Research Articles

Inhibition of p38 MAPK by SCIO-469 Suppresses TNF Generation and Promotes CD34+ Cell Survival in an In Vitro MDS Cell Culture Model.

Progress in the development of more effective therapeutics for myelodysplastic syndrome (MDS) has been limited by the lack of targets critical to the pathobiology of the disease. Ineffective hematopoiesis in MDS is characterized by accelerated proliferation and premature apoptotic death of progenitors and their progeny that is potentiated by the local generation of inhibitory molecules, including TNFa, TGFß, FasL, and VEGF. To identify upstream regulatory signals that may coordinate activation of inhibitory molecules, we used an in vitro cell culture model incorporating a CD34+ MDS cell line isolated from a RAEB-t patient, normal bone marrow stromal cells (BMSC), and/ or bone marrow mononuclear cells (BMMNC) to determine effects of cell-cell interactions on secretion of inhibitory hematopoietic cytokines. The role of p38 MAP kinase, a regulatory kinase involved in the convergence of inhibitory cytokine activation and signaling, was evaluated in this interaction. We found that p38 MAPK is induced under basal culture conditions in the MDS cell line and is further activated by TNFa or TGFß. In all cases, p38 activation was reduced by SCIO-469, a potent and specific inhibitor of p38a activity. SCIO-469 does not directly block p38 activation, suggesting a feedback loop is interrupted when p38 kinase activity is inhibited in MDS cells. To determine the effects of cellular interactions, the MDS cell line was co-cultured with either BMSC, BMMNCs or both from normal donors, and TNFa and FasL secretion were measured after 3 days incubation. TNFa and FasL were detected in culture supernatants when the MDS cell line was co-cultured with BMMNC but not when co-cultured with BMSC. TNFa secretion by BMMNCs was dependent on MDS cell contact and was significantly inhibited by SCIO-469. The addition of BMSC to the MDS and BMMNC co-culture prevented TNFa elevation, suggesting BMSCs as a dominant source for anti-inflammatory signal(s). VEGF, FGF-ß, TGFß2, BDNF, TIMP-1, TIMP-2 and IL-6 secretion by BMSC was induced by MDS co-culture, whereas SCIO-469 blocked cytokine induction. To determine the effects of SCIO-469 and MDS clone-induced BM cytokine secretion on normal CD34+ proliferation, we co-cultured BMMNCs and BMSC in transwell inserts in the presence or absence of the MDS cell line with or without SCIO-469. CD34+ proliferation was assessed in cells cultured in outer wells. CD34+ progenitors proliferated in culture at the same rate as those co-cultured with BMSC, BMMNC and MDS for 6 days. At longer intervals, viability of progenitors cultured with the MDS line declined, whereas treatment with SCIO-469 abrogated the decrease in CD34+ viability. These results implicate p38a as a critical target in the induction of pro-apoptotic cytokines in MDS, and that selective inhibition of p38 by SCIO-469 may provide a novel therapeutic strategy for MDS.

Cyclophilin a modulates dendritic cell differentiation from myeloblastic cell KG1

Introduction. Cyclophilin A (CypA) is a ubiquitously distributed intracellular protein as well as secreted protein and has recently been reported to be an immunomodulatory molecule. The objective of this study was to determine the effect of CypA on dendritic cell (DC) differentiation, activation, and functional maturation. Methods. KG1 cells (CD34 + human myeloblastic cell line) were treated with cytokines (GM-CSF+IL-4) and/or CypA and expression of cell-surface markers was analyzed by FACS analysis. The antigen uptake capacity of different DCs was determined by FITC-dextran uptake assay. Antigen presentation capacity of DCs was determined by allogeneic mixed lymphocyte reaction (MLR) by [ 3H]thymidine incorporation assay. To check the T cell polarization of KG1-derived DCs, various Th1 and Th2 cytokines secreted by allo-stimulated CD4 + and CD8 + T cells were determined by Bioplex cytokine assay. Role of p38 MAP kinase in DC functions was also investigated. Results. During the differentiation of KG1 cells to immature DCs, cell-surface expression of CD11b was increased by 30.6% for CypA alone; 55% for CypA plus cytokines; and 44% for cytokines alone. Similarly, CypA alone increased the cell-surface expression of CD11c by 59% as compared to CypA plus cytokines (68%) and cytokines alone (50%). CypA up regulated the antigen uptake capacity of the immature DCs to a greater extent (5 times) as compared to cytokines alone (2.5 times). Moreover, CypA augmented the capacity of DCs to present antigens to allogenic CD8 + T cells and also increased the secretion of Th1-type cytokines TNF-α and IFN-γ from the allogenic CD4 + T cells. Furthermore, CypA induced the phosphorylation and hence activation of MAP kinase p38. Pretreatment with SB-203580, a p38 inhibitor, significantly reduced MLR stimulatory capacity of CypA-induced DCs in both CD8 + T cells and CD4 + T cells ( P < 0.05). Conclusions. CypA enhances DC differentiation and maturation by up regulating CD11b and CD11c expression. CypA can also augment DC antigen uptake and antigen presentation, which may be mediated by the P38 signaling pathway.

The role of p38 MAP kinase in the synergistic cytotoxic action of calcitriol and TNF-α in human breast cancer cells

Calcitriol, the hormonal form of Vitamin D, potentiates the activity of some agents of the anti-cancer immune system including tumor necrosis factor-α (TNF-α). Different signaling pathways activated by TNF-α may be targets for calcitriol action. Activation of p38 MAP kinase was shown to have both pro- and anti-apoptotic actions in TNF-α-induced programmed cell death depending on cell context. Treatment of MCF-7 breast cancer cells with TNF-α resulted in activation of p38 MAP kinase that persisted for at least 24 h. Whereas calcitriol had no effect on the earlier phase of p38 MAP kinase activation (up to 1 h), it inhibited the activation of this pathway between one and 24 h after exposure to TNF-α. Both calcitriol and the p38 MAP kinase inhibitor SB203580 enhanced TNF-α-induced cytotoxicity and drop in mitochondrial membrane potential, but their combined effect was sub-additive. Taken together, these findings suggest that p38 MAP kinase plays an anti-apoptotic role in TNF-α-induced cytotoxicity in MCF-7 cells and that the synergistic interaction between TNF-α and calcitriol, leading to mitochondrial damage and subsequent cell death, is partially due to modulation of this signaling pathway.

Cell survival signalling in heart derived myofibroblasts induced by preconditioning and bradykinin: the role of p38 MAP kinase.

Fibroblasts possess receptors for compounds released during ischemia, including bradykinin. The aims of the present study were to investigate tyrosine kinase and p38 MAP kinase signalling in heart derived myofibroblasts in response to bradykinin and preconditioning ischemia. Fibroblasts from neonatal rat hearts were subjected to pharmacological agents and/or simulated ischemia. Cell viability was measured by the conversion of a tetrazolium salt to its formazan derivative. Preconditioning with 30 min of simulated ischemia followed by 30 min recovery resulted in an 85.4% +/- 7.8% increase in cell survival above that of cells treated with prolonged ischemia alone. Cells treated with bradykinin showed a 35% +/- 7.9 increase in cell survival after lethal ischemia. The B2 receptor antagonist Hoe 140 blocked the protective effect of bradykinin, but did not block preconditioning. The K(ATP) channel blocker glibenclamide and the mitochondria specific K(ATP) blocker 5, hydroxydecanoate, abolished the cytoprotection induced by both preconditioning and bradykinin. The non specific tyrosine kinase inhibitor genistein also abolished the cytoprotection. Effective blockade of cytoprotection was obtained with K(ATP) channel blockers and the tyrosine kinase inhibitor when these compounds were given prior to the preconditioning stimulus and not during the lethal insult. The stress activated protein kinase p38 MAP kinase was investigated by Western blotting and by the use of a specific inhibitor (SB203580). Preconditioning reduced phospho-p38 MAP kinase; in contrast, bradykinin administration markedly increased phosphorylation of p38 MAP kinase. SB203580 protected cells from lethal simulated ischemia. In conclusion, cell survival-signalling pathways activated by bradykinin or simulated ischemia in heart fibroblasts protect via the opening of K(ATP) channels and are independent of the stress-activated p38 MAP kinase and/or related to inhibition of this kinase.

An inhibitor of p38 MAP kinase downregulates cytokine release induced by sulfur mustard exposure in human epidermal keratinocytes

Sulfur mustard (2,2 ′-dichlorodiethyl sulfide, SM) is a potent alkylating agent that induces skin vessication after cutaneous exposure. Previous work has revealed that SM induces the production of inflammatory cytokines, including IL-8, IL-6, TNF-α, and IL-1β, in keratinocytes. The p38 MAP kinase (MAPK14) signaling pathway is activated via phosphorylation in response to cellular stress and has been implicated in the upregulation of cytokines in response to stress. We investigated the role of p38 MAP kinase in inflammatory cytokine upregulation following SM exposure. A dose response study in cultured human epidermal keratinocytes (HEK) revealed increasing phosphorylation of p38 MAP kinase in response to increasing concentrations of SM. A time course at the 200 μM exposure revealed that p38 MAP kinase phosphorylation is induced by 15 min post-exposure, peaks at 30 min and is sustained at peak levels until 8 h post-exposure. Phosphorylation of the upstream kinase MKK3/6 was also detected. Assay of the SM-exposed HEK culture media for cytokines revealed that exposure to 200 μM SM increased IL-8, IL-6, TNF-α, and IL-1β. When cells exposed to 200 μM SM were treated with the p38 MAP kinase inhibitor SB203580, the levels of IL-8, IL-6, and TNF-α and IL-1β were significantly decreased when compared with cells that were untreated. These results show that p38 MAP kinase plays a role in SM-induced cytokine production in HEK and suggest that inhibiting this pathway may alleviate the profound inflammatory response elicited by cutaneous SM exposure.

Moderate hypothermia during cardiopulmonary bypass (CPB) inhibits the intramyocardial synthesis of TNF-α: a role of p38 MAP kinase

Moderate hypothermia during cardiopulmonary bypass (CPB) inhibits the intramyocardial synthesis of TNF-α: a role of p38 MAP kinase

A role for p38 MAP kinase in platelet activation by von Willebrand factor.

Platelet activation induced by von Willebrand factor (VWF) binding to the membrane GPIb-IX-V receptor involves multiple signal transduction pathways. Among these, recruitment and activation of the FCgammaRIIA and stimulation of phospholipase A2 represent independent events equally essential to support a complete platelet response. Phospholipase A2 is activated by calcium and by phosphorylation through MAP kinases. In this work, we found that VWF stimulated the rapid and sustained phosphorylation of p38 MAP kinase (p38MAPK). In vitro kinase assay revealed that VWF-stimulated phosphorylation of p38MAPK was associated with increased kinase activity. Binding of VWF to GPIb-IX-V, but not to integrin alphaIibbeta3, was required to support phosphorylation of p38MAPK. Neither the blockade of the membrane FCgammaRIIA by a specific monoclonal antibody or the prevention of thromboxane A(2) synthesis by cyclooxygenase inhibitors affected VWF-induced p38MAPK activation. How-ever, phosphorylation of p38MAPK was prevented by the tyro-sine kinase Syk inhibitor piceatannol. Treatment of platelets with the p38MAPK inhibitor SB203580 totally prevented VWF-stimulated platelet aggregation. Moreover, release of arachidonic acid induced by VWF was strongly impaired by inhibition of p38MAPK. We also found that VWF induced phosphorylation of cytosolic phospholipase A(2), and that this process was prevented by the p38MAPK inhibitor SB203580. These results demon-strate that p38MAPK is a key element in the FCgammaRIIA-independent pathway for VWF-induced platelet activation, and is involved in the stimulation of phospholipase A(2) and arachidonic acid release.

Role of p38 MAP kinase in regulation of cell migration and proliferation in healing corneal epithelium.

The purpose of the present study was to examine the roles of signaling pathways potentially activated by TGFbeta (i.e., Smad and p38 mitogen-activated kinase [MAPK]) in regulation of cell migration and proliferation of healing mouse corneal epithelium. Activation of Smads or p38MAPK was evaluated by immunohistochemistry in healing mouse corneal epithelium after debridement. The role of endogenous TGFbeta or p38MAPK in epithelial healing was determined in organ-cultured mouse corneas with an epithelial defect, in the presence or absence of a TGFbeta-neutralizing antibody or p38MAPK inhibitors, respectively. Cell proliferation was evaluated by incorporation of bromodeoxyuridine. Migrating mouse corneal epithelium had minimal cell proliferation. Smad3 and -4 were found in nuclei of normal corneal epithelium, whereas they were absent in nuclei of migrating cells in association with Smad7 upregulation on epithelial debridement. Administration of TGFbeta-neutralizing antibody reduced the protein expression of Smad7 in vivo after a corneal injury. In contrast, phosphorylation and nuclear translocation of p38MAPK were markedly evident in migrating epithelium during healing, but not in uninjured epithelium. In organ culture, addition of p38MAPK inhibitors blocked cell migration more markedly than neutralizing TGFbeta-antibody and enhanced cell proliferation in the injured corneal epithelium, in association with phosphorylation of Erk. Endogenous TGFbeta enhances migration of corneal epithelium during wound healing in mice. The p38MAPK, but not the Smad, cascade plays a major role in promoting cell migration and in suppressing cell proliferation in migrating epithelium.

P38 MAP kinases: key signalling molecules as therapeutic targets for inflammatory diseases.

The p38 MAP kinases are a family of serine/threonine protein kinases that play important roles in cellular responses to external stress signals. Since their identification about 10 years ago, much has been learned of the activation and regulation of the p38 MAP kinase pathways. Inhibitors of two members of the p38 family have been shown to have anti-inflammatory effects in preclinical disease models, primarily through the inhibition of the expression of inflammatory mediators. Several promising compounds have also progressed to clinical trials. In this review, we provide an overview of the role of p38 MAP kinases in stress-activated pathways and the progress towards clinical development of p38 MAP kinase inhibitors in the treatment of inflammatory diseases.

Mechanisms of cardiac protection with Overexpression of A1 adenosine receptors

AbstractMyocardial damage from ischemia/reperfusion injury can be minimized through endogenous protective mechanisms, including activation of A1 adenosine receptors. Transgenic mice with cardiac‐specific A1 adenosine receptor overexpression (A1AR Trans) have demonstrated functional and metabolic tolerance to in vitro and in vivo myocardial ischemia/reperfusion injury. The purpose of this investigation is to examine the role of p38 MAP kinase in A1 receptor‐mediated cardioprotection. Activation of p38 MAPK by A1 adenosine receptor stimulation was evaluated in neonatal rat cardiomyocytes subjected to simulated ischemia. A1 activation by cyclopentyladenosine decreased cell death and simultaneously enhanced p38 phosphorylation by simulated ischemia. The role of p38 MAP kinase activation in cardioprotection with A1 adenosine receptor overexpression was evaluated in isolated perfused hearts from A1AR Trans mice subjected to 20 min ischemia and 30 min reperfusion. Inhibition of p38 MAPK activity with 10 μM SB‐203580 prior to ischemia and during reperfusion decreased postischemic total functional recovery in A1AR Trans hearts from 54 ± 1 to 27±7% of maximal and increased final EDP from 10±3 mmHg to 34±8 mmHg. A1 adenosine receptor‐mediated activation of p38 MAP kinase in cardiomyocytes is involved in the protection of cells from ischemic death. Tolerance to ischemic injury by A1 receptor overexpression is dependent upon activation of p38 MAPK. These findings suggest that adenosine A1 mediated cardioprotection in transgenic mice involves activation of the p38 MAP kinase cascade. Drug Dev. Res. 58:439–446, 2003. © 2003 Wiley‐Liss, Inc.

Potential of p38 MAP kinase inhibitors in the treatment of cancer.

The involvement of chronic inflammation in tumor development and progression is reviewed. Based on the natural history of certain diseases and epidemiology studies, a strong association has been established between particular chronic inflammatory conditions and eventual tumor appearance. Solid tumors require a stroma for their growth and recruit macrophages to synthesize essential growth and angiogenic factors that they do not have the capacity to produce. The microenvironment of the local host tissue appears to be an active participant in exchanging cytokines and enzymes with tumor cells that modify the local extracellular matrix, stimulate migration, and promote tumor angiogenesis, proliferation and survival. The role of p38 MAP kinase as a therapeutic target for treating cancer is discussed.

The Role of p38 MAP Kinase in Tumor Necrosis Factor-Induced Redistribution of Vascular Endothelial Cadherin and Increased Endothelial Permeability

The mechanism by which tumor necrosis factor alpha (TNFalpha) increases endothelial permeability is unclear. Vascular endothelial (VE) cadherin (cadherin 5) is an important contributor to endothelial monolayer integrity. The purpose of our study was to determine the effect of TNFalpha on VE-cadherin cell-surface expression and to identify the signaling pathways involved in TNF-induced changes in cadherin expression. Human umbilical vein endothelial cell monolayer permeability was measured by enzyme-linked immunosorbent assay for biotin-labeled albumin. Immunofluorescence, laser confocal microscopy, and Western immunobloting were used to assess VE cadherin distribution. Mitogen-activated protein kinase (MAPK) activity was determined using functional kinase assays and was inhibited with the compounds SB202190 and PD98059. TNFalpha significantly increased permeability and induced p38 and ERK MAPK activation compared with controls (P < 0.05). These changes were associated with a loss of membrane-associated VE cadherin. Inhibition of p38 but not ERK MAPK significantly reduced the effect of TNFalpha on endothelial permeability and cell-surface VE cadherin expression. p38 MAP kinase activation appears to be an important upstream signaling event associated with increased endothelial permeability and vascular endothelial cadherin redistribution.

The selective p38 inhibitor SB-239063 protects primary neurons from mild to moderate excitotoxic injury

Inhibition of the p38 mitogen-activated protein kinase (MAP Kinase) pathway reduces acute ischemic injury in vivo, suggesting a direct role for this signaling pathway in a number of neurodegenerative processes. The present study was designed to evaluate further the role of p38 MAP Kinase in acute excitotoxic neuronal injury using the selective p38 inhibitor SB-239063 ( trans-1-(4hydroxycyclohexyl)-4-(fluorophenyl)-5-(2-methoxy-pyrimidin-4-yl) imidazole). Unlike the widely used p38 inhibitor, SB-203580 (4-(4-Fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1 H-imidazole), this second generation p38 inhibitor more selectively inhibits p38 MAP Kinase without affecting the activity of other MAP Kinase signaling pathways and provides a more accurate means to selectively assess the role of p38 in excitotoxicity that has not been previously possible. SB-239063 provided substantial protection against cell death induced by either oxygen glucose deprivation (OGD) or magnesium deprivation in cultured neurons. The ability of this compound to block excitotoxicity was not due to direct inhibition of N-methyl- d-aspartate (NMDA) receptor-mediated currents as SB-239063 did not alter NMDA electrophysiological responses. SB-239063 did not protect against a severe excitotoxic insult induced by 60-min exposure to NMDA. However, when tested against a less severe, brief (5 min) NMDA exposure, p38 inhibition provided substantial protection. These data demonstrate that inhibition of p38 MAP Kinase can confer neuroprotection in vitro against mild but not severe excitotoxic exposure, and suggests that other additional pathways/mechanism(s) may be involved in severe excitotoxic cell death.

Farnesyl thiosalicylic acid inhibits the growth of melanoma cells through a combination of cytostatic and pro-apoptotic effects.

Novel classes of drug that interfere with the signalling of the small G-protein Ras, the so-called Ras antagonists, are showing much promise as novel anti-cancer agents. In this study, we demonstrate that the novel Ras antagonist farnesylthiosalicylic acid (FTS) inhibits the growth of Colo 853 melanoma cells through a combination of cytostatic and pro-apoptotic effects. Furthermore, these phenomena are seen under conditions of cell attachment and in the presence of serum. Treatment of Colo 853 cells with FTS led to time-dependent inhibition of constitutive Akt, retinoblastoma protein (pRB) and ERK activity, with a concurrent loss of Akt expression. Inhibition of Akt and ERK activity induces apoptosis in other human cancer cell lines. Here it is demonstrated that inhibition of Akt, or ERK and Akt in combination, leads to cell cycle arrest but not apoptosis in melanoma cells. FTS treatment was also found to upregulate activity of the stress-activated p38 MAP kinase. Inhibition of p38 MAP kinase, using the selective inhibitor SB 203580, followed by FTS treatment, significantly increased the proportion of apoptotic cells after 72 hr, possibly suggesting a modulatory role for p38 MAP kinase in FTS-induced melanoma cell apoptosis.

Evidence for a role of p38 MAP kinase in expression of alkaline phosphatase during osteoblastic cell differentiation

In the present study, we investigate the implication of the mitogen-activated protein kinases (MAPKs) Erk, p38, and JNK in mediating the effect of fetal calf serum (FCS) on the differentiation of MC3T3-E1 osteoblast-like cells. Erk is stimulated by FCS in proliferating, early-differentiating, as well as in mature cells. Activation of p38 by FCS is not detected in proliferating cells but is observed as the cells differentiate. JNK is activated in response to FCS throughout the entire differentiation process, but a maximal stimulation is observed in early differentiating cells. The roles of Erk and p38 pathways in mediating MC3T3-E1 cell differentiation was determined using specific inhibitors such as U0126 and SB203580, respectively. These experiments confirmed that the Erk pathway is essential for mediating cell proliferation in response to FCS, but indicated that this MAP kinase has little effect in regulating the differentiation of MC3T3-E1 cells. In contrast, p38 only marginally influenced proliferation, but appeared to be critical for the control of alkaline phosphatase (ALP) expression in differentiating cells. Finally, results obtained with high doses of SB203580, which also affected JNK activity, suggest that p38 and/or JNK are probably also involved in the control of type 1 collagen and osteocalcin expression in differentiating cells. The data indicate that MAPKs regulate different stages of MC3T3-E1 cell development in response to FCS. Distinct MAPK pathways seem to independently modulate osteoblastic cell proliferation and differentiation, with Erk playing an essential role in cell replication, whereas p38 is involved in the regulation of ALP expression during osteoblastic cell differentiation. JNK is also probably involved in the regulation of osteoblastic cell differentiation, but its precise role requires further investigation.

Role of p38 MAP kinase in the development of acute lung injury.

Acute lung injury (ALI) is characterized by an intense pulmonary inflammatory response, in which neutrophils play a central role. The p38 mitogen-activated protein kinase pathway is involved in the regulation of stress-induced cellular functions and appears to be important in modulating neutrophil activation, particularly in response to endotoxin. Although p38 has potent effects on neutrophil functions under in vitro conditions, there is relatively little information concerning the role of p38 in affecting neutrophil-driven inflammatory responses in vivo. To examine this issue, we treated mice with the p38 inhibitor SB203580 and then examined parameters of neutrophil activation and acute lung injury after hemorrhage or endotoxemia. Although p38 was activated in lung neutrophils after hemorrhage or endotoxemia, inhibition of p38 did not decrease neutrophil accumulation in the lungs or the development of lung edema under these conditions. Similarly, the increased production of proinflammatory cytokines and activation of NF-kappaB in lung neutrophils induced by hemorrhage or endotoxemia was not diminished by p38 inhibition. These results indicate that p38 does not have a central role in the development of ALI after either hemorrhage or endotoxemia.

Role of p38 MAP kinase in diperoxovanadate-induced phospholipase D activation in endothelial cells.

We previously demonstrated that diperoxovanadate (DPV), a synthetic peroxovanadium compound and cell-permeable oxidant that acts as a protein tyrosine phosphatase inhibitor and insulinomimetic, increased phospholipase D (PLD) activation in endothelial cells (ECs). In this report, the regulation of DPV-induced PLD activation by mitogen-activated protein kinases (MAPKs) was investigated. DPV activated extracellular signal-regulated kinase, c-Jun NH2-terminal kinase (JNK), and p38 MAPK in a dose- and time-dependent fashion. Treatment of ECs with p38 MAPK inhibitors SB-203580 and SB-202190 or transient transfection with a p38 dominant negative mutant mitigated the PLD activation by DPV but not by phorbol ester. SB-202190 blocked DPV-mediated p38 MAPK activity as determined by activated transcription factor-2 phosphorylation. Immunoprecipitation of PLD from EC lysates with PLD1 and PLD2 antibodies revealed both PLD isoforms associated with p38 MAPK. Similarly, PLD1 and PLD2 were detected in p38 immunoprecipitates from control and DPV-challenged ECs. Binding assays demonstrated interaction of glutathione S-transferase-p38 fusion protein with PLD1 and PLD2. Both PLD1 and PLD2 were phosphorylated by p38 MAPK in vitro, and DPV increased phosphorylation of PLD1 and PLD2 in vivo. However, phosphorylation of PLD by p38 failed to affect PLD activity in vitro. These results provide evidence for p38 MAPK-mediated regulation of PLD in ECs.

Role of p38 MAP kinases and ERK in mediating ultraviolet-B induced cyclooxygenase-2 gene expression in human keratinocytes.

The roles of p38 MAP kinases and ERK in UVB induced cox-2 gene expression were studied in a human keratinocyte cell line, HaCaT. UVB significantly increased cox-2 gene expression at both protein and mRNA levels. As we reported previously, p38 and ERK were significantly activated after UVB irradiation in HaCaT cells. In addition, treating the cells with p38 inhibitor SB202190 or MEK inhibitor PD98059 specifically inhibited UVB induced p38 or ERK activation, respectively. In this study, we further examined the roles of p38 and ERK in UVB induced cox-2 gene expression in HaCaT cells. We found that SB202190 strongly inhibited UVB induced COX-2 protein expression at different time points and various UVB doses. Furthermore, SB202190 markedly inhibited UVB induced cox-2 mRNA. Our data indicated that ERK did not play a role in UVB induced cox-2 gene expression in human keratinocytes since suppression of ERK did not significantly alter UVB induced increase of COX-2 protein and mRNA. These results suggested, for the first time, that activation of p38 is required for UVB induced cox-2 gene expression in human keratinocytes. Since cox-2 expression plays an important role in UV carcinogenesis, p38 could be a potential molecular target for chemoprevention of skin cancer.

Role of p38 MAP kinase in LPS-induced airway inflammation in the rat.

We investigated the effect of the p38 kinase inhibitor SB 203580 on airway inflammation induced by aerosolized lipopolysaccharide (LPS) in male Wistar rats. SB 203580 significantly inhibited (ED(50)=15.8 mg kg(-1)) plasma levels of TNF-alpha in rats challenged with LPS (1.5 mg kg(-1), i.p.). Aerosolized LPS induced a peak in TNF-alpha levels and the initiation of a neutrophilic response in bronchoalveolar lavage (BAL) fluid at the 2 h time point. Furthermore, the 4 h time point was associated with the peak in IL-1beta levels and the initial plateau of neutrophilia observed in the BAL fluid. SB 203580 (100 mg kg(-1)), had no effect on peak TNF-alpha levels or the associated neutrophilia in the BAL. Interestingly, the PDE 4 inhibitor RP 73401 (100 mg kg(-1)) significantly reduced both TNF-alpha levels and neutrophilic inflammation. However, the BAL fluid from rats pre-treated with either compound significantly inhibited TNF-alpha release from cultured human monocytes 18 h after LPS treatment (83.6 and 44.5% inhibition, respectively). Alternatively, SB 203580 (100 mg kg(-1)) produced dose-related inhibition of BAL IL-1beta levels (67.5% inhibition, P<0.01) and BAL neutrophilia (45.9% inhibition, P<0.01) 4 h after LPS challenge. P38 protein was present in lung tissue and the level of expression was not affected by LPS treatment. P38 kinase appears to be involved in the release of IL-1beta and the sustained neutrophilic response in the BAL fluid. This data may suggest a role for p38 inhibitors in the treatment of airway inflammatory diseases in which neutrophilia is a feature of the lung pathology.

Adenosine-induced late preconditioning in mouse hearts: role of p38 MAP kinase and mitochondrial K(ATP) channels.

We investigated the role of p38 mitogen-activated protein kinase (MAPK) phosphorylation and opening of the mitochondrial ATP-sensitive K(+) [(K(ATP))(mito)] channel in the adenosine A(1) receptor (A(1)AR)-induced delayed cardioprotective effect in the mouse heart. Adult male mice were treated with vehicle (5% DMSO) or the A(1)AR agonist 2-chloro-N(6)-cyclopentyladenosine (CCPA; 0.1 mg/kg ip). Twenty-four hours later, hearts were subjected to 30 min of global ischemia and 30 min of reperfusion in the Langendorff mode. Genistein or SB-203580 (1 mg/kg i.p.) given 30 min before CCPA treatment was used to block receptor tyrosine kinase or p38 MAPK phosphorylation, respectively. 5-Hydroxydecanoate (5-HD; 200 microM) was used to block (K(ATP))(mito) channels. CCPA produced marked improvement in left ventricular function, which was partially blocked by SB-203580 and 5-HD and completely abolished with genistein. CCPA caused a reduction in infarct size (12.0 +/- 2.0 vs. 30.3 +/- 3.0% in vehicle), which was blocked by genistein (29.4 +/- 2.3%), SB-203580 (28.3 +/- 2.6%), and 5-HD (33.9 +/- 2.4%). CCPA treatment also caused increased phosphorylation of p38 MAPK during ischemia, which was blocked by genistein, SB-203580, and 5-HD. The results suggest that A(1)AR-triggered delayed cardioprotection is mediated by p38 MAPK phosphorylation. Blockade of cardioprotection with 5-HD concomitant with decrease in p38 MAPK phosphorylation suggests a potential role of (K(ATP))(mito) channel opening in phosphorylation and ensuing the late preconditioning effect of A(1)AR.